Molecular phylogeny of the major arthropod groups indicates polyphyly of crustaceans and a new hypothesis for the origin of hexapods

A phylogeny of the arthropods was inferred from analyses of amino acid sequences derived from the nuclear genes encoding elongation factor-1 alpha and the largest subunit of RNA polymerase II using maximum- parsimony, neighbor-joining, and maximum-likelihood methods. Analyses of elongation factor-1 alpha from 17 arthropods and 4 outgroup taxa recovered many arthropod clades supported by previous morphological studies, including Diplopoda, Myriapoda, Insecta, Hexapoda, Branchiopoda (Crustacea), Araneae, Tetrapulmonata, Arachnida, Chelicerata, and Malacostraca (Crustacea). However, counter to previous studies, elongation factor-1 alpha placed Malacostraca as sister group to the other arthropods. Branchiopod crustaceans were found to be more closely related to hexapods and myriapods than to malacostracan crustaceans. Sequences for RNA polymerase II were obtained from 11 arthropod taxa and were analyzed separately and in combination with elongation factor-1 alpha. Results from these analyses were concordant with those derived from elongation factor-1 alpha alone and provided support for a Hexapoda/Branchiopoda clade, thus arguing against the monophyly of the traditionally defined Atelocerata (Hexapoda + Myriapoda).      

Toll-like receptor 7 and 8 polymorphisms: associations with functional effects and cellular and antibody responses to measles virus and vaccine

Successful defence against viral pathogens requires the rapid recognition of virus-specific “danger signals” and the activation of both innate and adaptive immunity. Toll-like receptors (TLR) 7 and 8 play a critical role in the elimination of viruses by recognising the common viral component, single stranded (ss)RNA. Measles virus, an ssRNA virus, continues to cause serious morbidity and mortality worldwide despite available measles vaccines. TLR7 and TLR8 genetic variation may cause functional alterations that result in impaired responses to measles. In a population of 12-month-old Australian infants, receptor protein expression was examined to assess the functionality of TLR7 and TLR8 polymorphisms, and the effects of these polymorphisms on cellular and antibody responses after the first measles vaccine dose were investigated. TLR7 Leu11Gln showed associations with TNF-α responses after ligand (imiquimod) stimulation in males only (P = 0.040), and non-responders were more likely to be Gln males (P = 0.044). TNF-α non-responders after imiquimod also had higher percentages of TLR8 -4284TT (69.6%) (P = 0.001) and TLR8 -558CC (69.6%) (P = 0.002) in females. Receptor protein expression after imiquimod or measles stimulation was not significantly altered compared with baseline, nor was it affected by genotype. None of the TLR7 or TLR8 polymorphisms studied were associated with measles-specific cytokine levels or with measles IgG levels. In conclusion, we report gender-specific associations with TLR7 and TLR8 polymorphisms and TNF-α cellular responses to its ligand. However, we found no evidence of any functional effects of TLR7 or TLR8 polymorphisms on receptor expression, measles-specific cellular responses or measles vaccine antibody responses.     

On the distribution of Na+ pump sites in the frog skin

Exposure of the outside of the isolated frog skin to a Ringer's solution, made hypertonic by the addition of mannitol, causes a rapid and sustained increase in transepithelial permeability through a structural distortion-a focal blistering-of the "tight" junctions of the outermost living cell layer. [(3)H]ouabain, used as an autoradiographic marker for the Na+-pump (Na+-K+-adenosine triphosphatase), is usually unable to penetrate the frog skin from the outside solution, but when added to a hypertonic mannitol- Ringer's solution in the outside bath it readily penetrates the epithelium, presumably through the opened shunt pathway. Radioautographic analysis of [(3)H]ouabain binding sites revealed that most of ouabain enters from the outside solution binds to the sites on the cell membranes of the stratum spinosum, as was the case when it was applied from the inside bath in an earlier study. The outer living cell layer, the first to be exposed to ouabain, does not appear to be the major site for the Na+-pump, and therefore, is not likely to be responsible for most of the active pumping of Na+. This result demonstrates that previous failure to show a high density of Na+-pump sites on the cells of the outermost layer, when [(3)H]ouabain was applied from the inside solution, was not due to the inability of the marker to reach these cells at a sufficient concentration to reveal all pump sites. These results provide further support for a model of Na+-transport across the frog skin which distributes the active pump step on the inward facing membranes of all living cells.     

Transposon tagging in diploid strawberry

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T(1) progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T(0) launch pads, putative transposants in the T(1) generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T(1) plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T(1) plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T(0) generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T(2) transposon-tagged plants. The mutant collection has been catalogued in an on-line database.     

Restriction-map variation with the yellow-achaete-scute region in five populations of Drosophila melanogaster

It has been proposed that the degree of recombination for a genomic region will affect the level of both nucleotide heterozygosity and the density of transposable elements. Both features of genomic diversity have been examined in a number of recent reports for regions undergoing relatively normal levels of recombination in Drosophila melanogaster. In this study the genomic variation associated with yellow-achaete- scute loci located at the tip of the X chromosome is examined by six- cutter restriction mapping. In this region, as usual for regions adjacent to telomeres, crossing-over is dramatically reduced, and published studies of visible mutants indicate extremely little restriction-map variation. Eight six-cutter restriction endonucleases were used to locate sequence variation in 14- and 16.5-kb regions in 109 lines sampled from North America, Africa, and Europe. The overall level of heterozygosity is estimated as 0.29%. Nine large insertions, all presumed to be transposable elements, were observed. Base-pair heterozygosity appears to be reduced compared with regions having normal levels of recombination. The estimated heterozygosity is much higher than reported in earlier studies of restriction-map variation among visible mutations in the complex. The incidence of large insertions is not elevated compared with that in other regions of the genome. This suggests that asymmetric synapsis and exchange is not an important mechanism for the elimination of transposable elements.      

Oxidative stress in liver and brain of the hatchling chicken (Gallus domesticus) following in ovo injection with TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was injected into chicken eggs prior to incubation to study possible mechanisms of toxicity and teratogenicity. One of the suggested mechanisms of teratogenicity is oxidative stress. Eggs were injected simultaneously with TCDD and cotreatment compounds in an attempt to prevent oxidative stress or to block cytochrome P450 activity. Indicators of oxidative stress were assessed in livers and brains of hatchling chicks. In ovo, exposure to TCDD caused significant effects on indicators of oxidative stress in liver, but not in the brain of the hatchling chicks. TCDD did not significantly affect superoxide production. In liver, TCDD treatment caused a decrease in glutathione content and glutathione peroxidase activity and an increase in the ratio of oxidized to reduced glutathione. TCDD increased the susceptibility to lipid peroxidation and oxidative DNA damage in liver. Administration of the antioxidants vitamin E and vitamin A provided partial protection against TCDD-induced oxidative stress in liver. The lack of effect of TCDD in chicken brain could be due to the low cytochrome P4501A activity in this tissue and little accumulation of TCDD in brain compared to liver. Phenytoin, a known inducer of oxidative stress, caused a decrease in glutathione content and an increase in susceptibility to lipid peroxidation in both liver and brain and increased oxidative DNA damage in brain. Responsiveness varied among individual animals, but measures of the oxidative stress were correlated.     

Photooxidative reactions in chloroplast thylakoids. Evidence for a Fenton-type reaction promoted by superoxide or ascorbate

A methyl viologen (MV)^* mediated Mehler reaction was studied using Type C and D chloroplasts (thylakoids) from spinach. The extent of photooxidative reactions were measured as (a) rate of ethylene formation from methional oxidation indicating the production of oxygen radicals, and (b) rate of malondialdehyde (MDA) formation as a measure of lipid peroxidation. Without added ascorbate, 1 M FerricEDTA increased ethylene formation by greater than 4-fold, but had no effect on MDA production. Ascorbate (1 mM) produced a tripling of ethylene while it reduced MDA formation in the presence of iron. Radical scavengers diethyldithiocarbamate (DDTC), formate, 1,4-diazabicyclo (2.2.2octane) (DABCO), inhibited ethylene formation. Using 0,4 M mannitol to scavenge hydroxyl radicals, the rates of ethylene formation were reduced 40 to 60% with or without 1 M Fe(III) EDTA. The strong oxidant(s) not scavenged by mannitol are hypothesized to be either alkoxyl radicals from lipid peroxidation, or site specific formation of hydroxyl radicals in a lipophillic environment not exposed to mannitol. Singlet oxygen does not appear to be a significant factor in this system. Catalase strongly inhibited both ethylene and MDA synthesis under all conditions; 1 mM ascorbate did not reverse this inhibition. However, the strong superoxide dismutase (SOD) inhibition of ethylene and MDA formation was completely reversed by 1 mM ascorbate. This suggests that superoxide was functioning as an iron reducing agent and that in its absence, ascorbate was similarly promoting oxidations. Therefore, these oxidative processes were dependent on the presence of H₂O₂ and a reducing agent, suggesting the involvement of a Fenton-type reaction.Abbreviation DABCO 1,4-diazabicyclo(2.2.2.octane) - DCMU 3-(3,4 Dichlorophenyl). 1,1-dimethyl urea - DDTC diethyldithiocarbamate - EDTA ethylenediamine-tetraacetic acid - MDA malondialdehyde - MV methyl viologen - SOD superoxide dismutase - TBA thiobarbituric acid - TCA trichloroacetic acid Scientific contribution number 1315 from the New Hampshire Agriculture Experiment Station.